Throughout this application, various publications are referenced by Arabic numerals within parentheses. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
The phylum Apicomplexa includes hundreds of different organisms belonging to the order Eucoccidiorida. The genus Eimeria is included within the order of true coccidian agents. Of the organisms belonging to this genus, several species are of recognized importance to the chicken industry. These species include Eimeria tenella, E. maxima, E. acervulina, E. necatrix, E. brunetti, E. mivati, E. mitis and E. praecox.
Differentiation of species is based on the site of infection within the host and oocyst morphology. To date, biochemical markers have not been used for speciation, although differences have been noted for each of the above species.
For avian Eimeria, the entire life cycle is completed within a single host. The life cycle is complex consisting of asexual and sexual stages depending upon the Eimeria species involved. The infective stage is the sporulated oocyst. Upon being ingested in contaminated feces, food or water, sporulated oocysts excyst within the digestive tract as a result of the combined action of mechanical shearing and enzymatic hydrolysis of the sporocyst cap. The liberated sporozoites traverse epithelial cells within specific regions of the intestine.
Development begins within the Crypt of Lieberkuhn to the level of first generation meronts; the meront is a transitional stage consisting of rounded organisms with a more pronounced nucleus, plus increased energy generating and protein synthesizing capacity. Development of first-generation merozoites follows due to multiple fission of meronts. The release of first-generation merozoites destroys the host cell, and the parasites migrate to infect new host cells undergoing a second asexual cycle. Meronts develop to the level of second-generation merozoites destroying additional epithelial cells as they are released. Further destruction of host cells follows with the liberation of the third-generation merozoites. The number of merozoite generations varies from one Eimeria species to another.
Sexual development commences with the production of microgametes and macrogametes through the process of gametogenesis. Liberated microgametes fertilize macrogametes to form zygotes. Development of immature oocysts is followed by rupture of the host cell. Oocysts, released into the lumen of the gut, are passed through the feces to the environment and mature (sporulate) in the presence of atmospheric oxygen.
The process of parasite development is self-limiting if the host ingests no additional oocysts. However, this proves to be an unrealistic expectation in crowded poultry houses.
Disease due to Eimeria can result in severe economic losses associated with diminished feed efficiency and pathologic manifestations.
The pathology of coccidiosis due to E. tenella and E. necatrix is in large part related to the rupture of host cells during the release of merozoites, while host cell rupture during the release of E. maxima oocysts contributes to the pathology seen with that species. Bleeding within the gut is related to rupture of small capillaries servicing the epithelium. It may be difficult to control the progress of disease using coccidiostats, once asexual development is established. Secondary infection often complicates the disease caused by Eimeria. Death can occcur within 4-7 days in infected birds infected with E. tenella or E. necatrix. However, death rarely occurs as a result of infection by E. maxima.
A consistent property of the coccidia is that the sporozoites initiate the infection process within very specific tissue sites (39, 45, 57). The site specificity of infection is a characteristic commonly used for speciation of Eimeria. For example, the asexual stages of E. necatrix show a propensity for invasion of epithelial cells residing within the mid-intestine, while sexual stages develop primarily in the cecal pouches.
Much of the work on immunity to coccidiosis has been confined to humoral immunity, more specifically to serum antibodies. Studies have shown a lack of correlation between serum antibody and resistance to disease (59). However, most available data support the contention that a local response with involvement of the secretory immune system or cell mediated immunity (CMI), or both, are involved in the protective response.
Interference with recognition, penetration and/or attachment of pathogens to host cells has a demonstrated protective effect as shown with viral, bacterial and protozoan models. Genetic deletion of key host cell receptors or pathogen attachment features can prevent the initial colonization process (16, 54). Alternatively, secretory antibodies can interfere with the colonization process by binding to, and consequently masking requisite receptors (32, 74). More than one immunoglobulin class has been reported to have the capacity of interfering with the initial colonization process of Eimeria tenella (13). However, recent reports indicate that only production of secretory IgA has been correlated with natural protective immunity (12, 59). Porter and Davis (13) and others (59) reported that secretory IgA neutralizes the extracellular stages of the parasite either by significantly limiting penetration or so debilitating those organisms which did penetrate as to prevent subsequent development.
It has been estimated that an amount approaching $0.5-1.0 billion is spent annually by producers worldwide to combat disease, or to curb the devastating effect of coccidiosis in chickens (39, 52). Even with control measures currently in use, poultry losses are substantial with estimates in the multi-million dollar range (77).
Currently, the most widely used means of controlling Eimeria in chickens is through the application of antiprotozoal chemical feed additives. The specific composition varies with the coccidiostat used, and each product affects only certain stages of the coccidian life cycle (39, 51, 58). Disadvantages of using coccidiostats are many, including short-term residual protection in birds, occasional diminished performance, invocation of resistance to the drug in parasites, and to some extent, safety. Products currently remain on the market for only a few years because of the development of drug resistant strains. This adds considerable pressure on the cost of development and continued manufacture of efficacious products (51).
Protection of birds by immunization has met with some success. Investigators have been able to invoke limited protection using preparations of killed organisms (1, 41, 43). A more effective approach for immunization of chickens has been with the use of a live protozoal product--e.g. Coccivac.TM. (15). The product, being a multivalent composition containing low doses of viable oocysts, is administered in drinking water to invoke a mild parasitemia in birds. A drawback of this product has been occasional depressed performance of birds during the first weeks following administration. Variables such as excessive dosing or moisture content of bedding have even led to severe outbreaks of coccidiosis. See also, U.S. Pat. No. 3,147,186 (1964) which concerns the use of viable, sporulated oocysts of E. tenella to immunize chickens and U.S. Pat. No. 4,301,148 (1981) which concerns the use of sporozoites of E. tenella for the same purpose.
An alternative means of introducing the live vaccine into broiler houses is by way of the feed. This has been considered in a recent British patent (GB2,008,404A). Prior to mixing with the feed, fully virulent oocysts of E. tenella are encapsulated in a water soluble polysaccharide to protect against desiccation. The oocysts are in sufficient amounts only to induce subclinical infection. Though the immunizing ability was found to be excellent, no development of this method is foreseen due to questionable field acceptability. However, if attenuated strains of all the important coccidia could be developed, the procedure may be more acceptable.
Efforts have indeed been made to develop Eimeria lines of reduced virulence. Some species have been successfully attenuated through chicken embryo passage (19, 37, 40, 66). These strains have diminished ability to cause disease, yet have retained sufficient immunogenicity to invoke immunity. Some problems do, however, remain with the handling of these strains. As examples, the attenuated variants of E. necatrix have a critical passage limit whereby more or less embryo passage can result in loss of immunogenicity or maintenance of the original virulent form. Furthermore, some attenuated organisms revert to the virulent form upon minimal back-passage through chickens (38, 68). Thus, problems associated with maintaining consistent properties in attenuated organisms are apparent.
Attenuation by precocious selection has also been practiced when Eimeria strains cannot be readily passaged through embryonated eggs. In this process, shed oocysts are harvested late in the prepatent period prior to the onset of heavy oocysts shedding (28, 48, 50, 67). Such selection results in cultures having abbreviated life cycles, and a corresponding diminution in virulence properties (28, 48, 50, 67). Though the trait of precocity for E. tenella (29) and E. acervulina (49) has been demonstrated to be genetically stable, not enough information is known about this method to assess its usefulness as a tool in the poultry industry.
There is little information available about the surface antigen composition of avian coccidia. Hybridoma cell lines which secrete monoclonal antibodies directed to antigens on the surface of sporozoites of Eimeria tenella have been reported (82). The antigens were not identified, other than that their molecular weights were between 13 and 150 kilodaltons. Additionally, no biological significance or described efficacy in a vaccine was attributed to the antigens. European Patent Publication No. 135,712 also discloses monoclonal antibodies which react with sporozoites of E. tenella. E. tenella sporozoite antigens are disclosed by this publication. Furthermore, European Patent Publication No. 135,073, corresponding to U.S. Pat. No. 4,650,676, discloses monoclonal antibodies which react specifically against merozoites and sporozoites of E. tenella. Merozoite antigens derived from E. tenella are described.
Previous work in the laboratory of M. H. Wisher suggests the presence of approximately 16 polypeptides identified by surface iodination of excysted sporozoites of E. tenella and having molecular weights form 20,000 to greater than 200,000 (81). Additionally, European Patent Publication No. 167,443 discloses extracts from sporozoitesor sporulated oocysts of E. tenella which may be used as vaccines to protect against coccidiosis. These extracts contain a plurality of polypeptides, one or more of which may be used as an antigen to protect against coccidiosis. Moreover, International Publication No. WO/00528 discloses a cloned gene or fragment thereof from E. tenella which encodes antigenic proteins. These proteins bind with a monoclonal or polyvalent antibody directed against an antigenic protein of avian coccidia.
Subunit approaches to vaccine development have proven successful over the past few years. In such approaches, candidate protective antigens are identified and characterized for the purpose of eventual preparation on a large scale. In studying parasite antigens, one research group used monoclonal antibodies to identify a potential protective antigen on the surface of Babesia bovis (83). A B. bovis antigen of 44,000 daltons has been identified, which when purified and injected into experimental animals afforded some level of protection against primary challenge. An immunologically important 30,000 dalton protein of Toxoplasma gondii has also been identified using monoclonal antibodies (31).
Since mid-1981, Danforth and coworkers have published several papers in which they indicate the possibility of producing monoclonal antibodies toward antigens of avian Eimeria species (9, 10, 11). Similarly, Speer, et al. (69, 70) have demonstrated the development of hybridomas against E. tenella and some physiologic properties thereof. Antibody-secreting hybridomas have been selected on the basis of an indirect fluorescent antibody test (10). The patterns of reaction, as observed with ultraviolet microscopy, have varied depending upon the monoclonal antibody used. Patterns have included exclusive reaction with sporozoites only vs reaction with sporozoites and merozoites; staining of the anterior portion of the sporozoite vs the entire membrane; and staining of distinct internal organelles vs non-descript internal staining (11).
Although the preparation of murine-origin hybridomas producing monoclonal antibodies is commonly practiced by those familiar with the art, there is nothing to suggest that the direct and specific selection of sporozoite-neutralizing hybridomas against the species E. tenella and E. necatrix or merozoite-neutralizing hybridomas against the species E. maxima will subsequently identify virulence determinants of these species which may be useful in the development of a subunit vaccine.
This invention concerns the identification, characterization, preparation and use of polypeptide antigens for development of immunity to coccidiosis caused by E. tenella, E. necatrix and E. maxima. Recombinant polypeptide antigens, including fusion proteins, are also described.
The antigens are capable of being precisely dispensed interms of direct antigenic content and cannot cause disease thus avoiding vaccine strain-related outbreaks and reversions or changes in immunologic properties.
Due to the large economic losses caused by coccidiosis in chickens, vaccines against E. tenella, E. necatrix and E. maxima are desirable. Using hybridoma technology, applicants have identified and purified potential protective antigens for use in subunit vaccines. Use of such a subunit vaccine avoids vaccine strain-related outbreaks and reversions or changes in immunological properties associated with the use of a live vaccine.
The quantity of parasite antigens that can be prepared from the organism is quite low and very costly. Recombinant DNA cloning and expression techniques have opened up a new approach to producing large amounts of protective antigens inexpensively. In simplest terms, these techniques require that DNA sequences encoding all or part of the antigen be placed in a cell, under the control of the genetic information necessary to produce the antigenic protein in that cell. The genetic information may be synthetic DNA (17), genomic (e.g., viral) or chromosomal DNA, or cDNA made from the mRNA encoding the antigen. The latter approach is the most direct method for complex organisms such as Eimeria sp.
However, because the cDNA only contains genetic information corresponding to the amino acid sequence of the antigen, it must be inserted into expression vectors that provide the genetic signals necessary for expression of the cDNA gene (i.e., transcription and translation). The antigens can be synthesized either alone or as products fused to another protein in E. coli.
Production of an effective subunit vaccine in E. coli has been reported for foot and mouth disease virus of swine and cattle (33, 66). Foot and mouth disease virus surface antigens were produced as fusion protein antigens in E. coli. Significant levels of virus-neutralizing antibody were raised when cattle and swine were immunized with these antigens. The recombinant DNA-derived antigens gave protection against challenge with foot and mouth disease virus.
In contrast to simple organisms such as foot and mouth disease virus where the genome and surface proteins have been studied extensively, very little is known about the molecular biology of Eimeria. Wang and Stotish (79, 80) reported rapid but transient RNA and protein synthesis in E. tenella during the first 6-8 hours after initiation of sporulation and suggested that all protein and nucleic acid synthesis during sporulation occurs in these first few hours. For example, Stotish et al. (72) reported a 30,000 dalton glycoprotein protein component of sporozoite membranes that was synthesized by unsporulated oocysts and later incorporated into sporozoite membranes during the process of sporulation. Recently, Stotish et al. (73) reported isolation and in vitro translation of RNA from unsporulated oocysts, oocysts during sporulation and from sporozoites. The in vitro translation products ranged from less than 10,000 daltons to greater than 200,000 daltons. Patterns for unsporulated and sporulating oocyst RNA directed-protein synthesis were different, suggesting that different RNA populations may exist during sporulation.
In order to produce cDNA encoding the antigenic proteins, it was necessary to determine when the mRNA encoding the antigenic proteins occurred during the life cycle of E. tenella. This invention concerns the isolation and characterization of cDNA clones encoding antigenic proteins and the production of engineered antigenic proteins in E. coli. It also concerns the extraction of these proteins produced in E. coli from the insoluble state and the process to make the proteins immunoreactive with monoclonal antibodies. Finally, this invention shows the preparation and use of the bacterially produced antigenic proteins to produce immunity in chickens to coccidiosis caused by E. tenella, E. necatrix and E. maxima.
Antigenic proteins derived from Eimeria tenella and vaccines containing them for the prevention of coccidiosis caused by E. tenella have been described in European Patent Publication No. 164,176.